Whole Genome Sequencing of a Citrobacter freundii Strain Isolated from the Hospital Environment: An Extremely Multiresistant NDM-1 and VIM-48 Coproducing Isolate

Abstract

Citrobacter freundii has acquired resistance to several antimicrobial drugs, including last-resort antibiotics affecting, therefore, clinical efficacy and causing high rates of mortality. In this study, we investigate the whole genome sequence of a carbapenem-resistant C. freundii strain isolated from the hospital environment in Tunisia. A total of 210 samples were taken using sterile swabs, from inanimate surfaces, medical devices, and care staff, during the period extended between March and April 2019. After the microbiological analysis of samples and antimicrobial susceptibility testing, only one strain identified as C. freundii showing resistance to carbapenems was selected for the whole genome sequencing. The genome analysis revealed a high-level resistance to most antibiotics. Interestingly, we have noted the coexistence of bla_NDM-1 and bla_VIM-48 metallo-β-lactamase (MBL) encoding genes conferring resistance to carbapenems. Other β-lactamases encoding genes have also been detected, including bla_TEM-1, bla_CMY-48, and bla_OXA-1. Moreover, genes conferring resistance to aminoglycoside [aac(3)-IId, ant(3″)-Ia, aadA, aac(6′)-Ib], macrolide [mph(A)], sulfonamide (sul1), trimethoprim (dfrA1), tetracycline [tet(D)], chloramphenicol [cat(B3)], rifamycin (arr-3), and quinolone (qnrB) have been revealed. The multi-locus sequence typing analysis showed that this isolate could not be assigned to an existing sequence type (ST), but it is almost identical to ST22. The plasmid investigation revealed the presence of five plasmids belonging to diverse incompatibility groups (IncFII, IncHI1A, IncHI1B, IncN, and IncX3). To the best of our knowledge, our findings report the first detection of NDM-1 and VIM-48 coproducing C. freundii in Tunisia and the second detection in the world of the bla_VIM-48.

Introduction

Citrobacter freundii is an opportunistic pathogen previously recognized as an environmental contaminant.¹ This species is now involved in several nosocomial and community infections such as neonatal meningitis, pneumonia, gastroenteritis, and urinary tract infections.^2–4 Because of the high selection pressures in the hospital environment, C. freundii can acquire several resistance genes, constituting, therefore, an important source of hospital infections.^5,6 This multiresistance is an emerging threat that could eventually lead to therapeutic deadlocks especially after the emergence of bacteria resistant to last-resort antibiotics such as the case of carbapenems.⁷

This class includes antibiotics with large spectrum bactericidal activity and is kept as a last resort for the treatment of infections by multidrug-resistant bacteria.⁸ They are effective against most Enterobacteriaceae species, including those that produce an ampC β-lactamases and extended-spectrum-β-lactamases. In fact, they are often used for the treatment of meningitis, pneumonia, intra-abdominal infections, gynecological infections, diabetic foot infection, and suspected or proven nosocomial infections with resistant gram-negative bacteria.^9–12

Unfortunately, the immoderate use of these antibiotics in the treatment of severe infections has led to the spread of resistant strains mainly through their acquisition of carbapenemase genes.¹³ Several carbapenemase genes have so far been detected worldwide with a predominance of KPC, IMP, VIM, NDM, and OXA48 encoding genes and this still depends on species, epidemiological status, therapeutic and surveillance strategies in countries and regions.^13–15 Carbapenemase producing C. freundii still scarcely reported not only on the regional level, but also on the continental or even on the global level. In Tunisia, it has been reported that the most common species acquiring carbapenemase encoding genes were Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa.¹⁶ Only one carbapenemase producing C. freundii strain has been hitherto detected in Tunisia.^16,17

Many carbapenemases are encoded on plasmids, which facilitate the spread of resistance genes among microorganisms of the same species or even among different bacterial species.¹⁸ In this study, we investigate the whole genome sequence of a carbapenem-resistant C. freundii isolated from the environment of a university hospital in Tunisia.

Materials and Methods

Samples

A total of 210 samples were taken using sterile swabs, from inanimate surfaces (doorknobs, beds, treatment tables, sinks, and others) medical devices (oxygen masks, thermometers, catheters, and others) and care staff (hands and blouses), from various services (obstetric and gynecology service, orthopedics and trauma service, catheterization room, internal medicine service, neonatology service, cardiovascular and thoracic surgery service, and hematology service) of the Military hospital of Tunis during the period extended between March and April 2019.

Bacterial isolation and selection of carbapenem-resistant isolates

Samples were inoculated on MacConkey agar (Biolife, Milan, Italy), and then incubated at 37°C for 24 hours. This selective medium allows the growth and differentiation of gram negative bacilli based on morphological and biochemical characteristics. The next step in this study was the selection of isolates showing resistance to one or more carbapenem antibiotics to perform the whole genome sequencing, thus allowing molecular identification and full characterization of resistance genes. Isolates were, therefore, tested by Kirby–Bauer disk diffusion method for susceptibility to three antibiotics of carbapenem class (meropenem, ertapenem, and imipinem) to select carbapenem-resistant strains.

The results were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST).¹⁹ The selected carbapenem-resistant strain was tested for 13 other antimicrobial agents, including ticarcillin, piperacillin/tazobactam, cefalexin, cefoxitin, cefotaxim, ceftazidim, amikacin, gentamicin, ciprofloxacin, tigecycline, fosfomycin, colistin, and trimethoprim sulfamethoxazole, by measuring the minimum inhibitory concentration (MIC) by Vitek II compact^® (BioMérieux^®) automated system using the AST-N357 card.

Next-generation sequencing

DNA isolation from the bacterial sample was carried out using the automated nucleic acid isolation system (QIAcube; Qiagen) and then measured using Qubit (Thermo Fisher Scientific). The creation of DNA libraries was done according the following steps: first, nucleic acids were fragmented by sonication method, and in the next stage, DNA fragments were labeled with the Nextera^™ DNA Flex Library Prep (Illumina) kit in accordance with the kit manufacturer's instructions. The concentrations of the prepared library were measured fluorometrically using the Qubit device according to the manufacturer's instructions in Thermo Fisher Scientific. The final concentration of the library was brought to 2 nM concentration and was loaded into the Illumina MiniSeq device according to the manufacturer's instructions and next-generation DNA sequence data were obtained.

Bioinformatic analysis

Antimicrobial resistance genes were analyzed using ResFinder3.1²⁰ and Kmer Resistance 2.2 server systems,²¹ the sequence type (ST) was determined with the multi-locus sequence typing (MLST) database and the existing plasmid incompatibility types were investigated using PlasmidFinder server system.²²

Results

Our study showed that only one carbapenem-resistant strain identified as C. freundii was isolated from a total of 210 samples taken from inanimate surfaces, medical devices, and care staff from various services of the military hospital of Tunis.

This isolate was detected in the sink of the internal medicine service and showed resistance to the three tested antibiotic of carbapenem class: ertapenem, meropenem, and imipenem. In addition to its resistance to carbapenems, this strain was extremely resistant to many antibiotics, including ticarcillin ([MIC] > 128 μg/mL), piperacillin/tazobactam ([MIC] > 128 μg/mL), cefalexin ([MIC] > 64 μg/mL), cefoxitin ([MIC] > 64 μg/mL), cefotaxim ([MIC] = 32 μg/mL), ceftazidim ([MIC] > 64 μg/mL), amikacin ([MIC] > 16 μg/mL), gentamicin ([MIC] > 16 μg/mL), ciprofloxacin ([MIC] > 16 μg/mL), trimethoprim sulfamethoxazole ([MIC] > 320 μg/mL). It seems that only three antibiotics including, tigecycline ([MIC] < 2 μg/mL), fosfomycin ([MIC] < 16 μg/mL), and colistin ([MIC] < 0.5 μg/mL), remain effective against this strain.

The whole genome analysis of C. freundii isolate revealed the existence of several resistance genes that were detected using ResFinder server system. Interestingly, we have noted the presence of two metallo-β-lactamase (MBL) genes: bla_NDM-1 and bla_VIM-48 that confer high-level resistance to carbapenems. The coexistence of two MBL genes was very scarcely reported worldwide. Other β-lactamases encoding genes have also been detected, including bla_TEM-1, bla_CMY-48, and bla_OXA-1.

In addition to its resistance to β-lactams, this strain was resistant to other antibiotic families, including aminoglycosides, macrolides, tetracyclines, sulfonamides, quinolones, phenicols, and rifamycins. In fact, the genome analysis of this multiresistant isolate revealed the presence of aac (3)-IId, ant (3″)-Ia, aadA, and aac (6′)-Ib genes, which confer resistance to gentamicin, streptomycin, tobramycin, and amikacin, respectively. Genes conferring resistance to macrolide [mph(A)], sulfonamide (sul1), trimethoprim (dfrA1), tetracycline [tet(D)], chloramphenicol [cat(B3)], rifamycin (arr-3), and quinolone (qnrB) have also been revealed.

The MLST analysis revealed the following allele combination: 100% arcA (5), 97% aspC (15), 92%clpX (12), 100% dnaG (11), 100% fadD (20), 100% lysP (17), and 100% mdh (15). Thus, this isolate could not be assigned to an existing ST, but it is almost identical to ST22 (a little difference has observed in aspC and clpX alleles).

The plasmid investigation revealed the presence of five replicon plasmids belonging to diverse incompatibility groups (IncFII, IncHI1A, IncHI1B, IncN, and IncX3). Actually, most of the acquired resistance genes are mobilized by these plasmids that favor the spread of these resistance genes between various species.¹⁶

Discussion

C. freundii was seldom studied not only in Tunisia but also all over the world and has usually been considered as an environmental contaminant.¹ Nevertheless, with the general increase of antimicrobial resistance, it has been reported that multiresistant C. freundii has been responsible for severe infections in neonates and in immunocompromised patients with therapeutic impasses.^23,24

In our study, we report the detection in the hospital environment of a multiresistant C. freundii coharboring two MBL genes: bla_NDM-1 and bla_VIM-48. To the best of our knowledge, this is the first report in Tunisia of the coexistence of two MBL genes in C. freundii. The coproduction of two metallo-enzymes in Enterobacteriaceae species has been scarcely reported and it has often been observed in K. pneumoniae. In our previous study, we have reported the first detection of VIM-1 and NDM-1 coproducing K. pneumoniae strain isolated from the blood of a patient hospitalized in the intensive care unit in the Military hospital in Tunis.²⁵

To date, only one study has reported the coproduction of two MBL by an extremely drug-resistant C. freundii strain isolated from an 18-year-old French patient returning from India and suffering from cerebrovascular malformation.²⁶ Whereas various MBL genes, including bla_NDM-1, bla_VIM-1, bla_VIM-4, and bla_IMP-8, in association with other carbapenemase genes (non-MBL) particularly bla_OXA-48 and bla_KPC-2 have been detected in C. freundii.^27–30 Likewise, we report in our study the second detection in the world of the bla_VIM-48 gene, which was first found in Pseudomonas putida isolated from chicken in China.³¹ It has been reported that VIM-48 increases the enzymatic activity, thermostability, and resistance to carbapenems.³²

Usually, the presence of two genes conferring resistance to the same antibiotic could influence the minimum inhibitory concentration (MIC), it increases, therefore, the level of resistance that makes the treatment more difficult or even impossible especially in the case of resistance to last-resort antibiotics.³³ In our study, this strain contained two metallo-β-lactamases, which could increase the level of resistance to carbapenems. The worst is that this strain was resistant to the most antibiotics, including the last-resort ones.

It is noteworthy that the strain included in this study produced all β-lactamase classes. In fact, in addition to its production of Ambler class B β-lactamases, this strain contained bla_TEM, bla_CMY-48, and bla_OXA-₁ genes that confer its production of Ambler class A, class C, and class D β-lactamases, respectively. Regarding the bla_CMY-48 gene, a subvariant of the bla_CMY-2 gene was naturally present in C. freundii.³⁴ This strain was also resistant to several antibiotic families, including aminoglycosides, macrolides, sulfonamides, trimethoprims, tetracyclines, chloramphenicols, rifamycins, and quinolones showing, therefore, that this isolate was extremely multidrug resistant and that the hospital environment could be a reservoir of multiresistant bacteria, thus constituting a high risk of nosocomial infections.^35–39

The plasmid investigation revealed the presence of five plasmids belonging to various incompatibility groups (IncFII, IncHI1A, IncHI1B, IncN, and IncX3) showing, therefore, that the resistance determinants can be mobilized on plasmids that constitute a reservoir of acquired resistance genes that could be transmitted to other species. The coexistence of five different resistance plasmids has been recently reported in a C. freundii strain isolated from a urine specimen from an esophageal cancer patient with hospital-acquired postoperative urinary tract infection from a teaching hospital in Chongqing City, China.⁴⁰ In this Chinese investigation, authors have showed that the coexistence of multiple plasmids and an extremely large number of resistance genes in a single bacterial isolate reflect active and complex horizontal genetic transfer events that have taken place under selective pressures associated with many kinds of antibiotic molecules or toxic compounds.⁴⁰

In our study, although samples were taken after surface disinfection, this strain has persisted in the hospital environment. This could be related to several factors, including the ability of this strain to adapt to selective pressures, such as antimicrobials and biocides present in the hospital environment. The MLST analysis showed that the ST of this C. freundii is very similar to ST22. Hence, it is likely that this strain has derived from ST22 clone. A recent article focusing on the global evolutionary epidemiology and resistome dynamics of seldom isolated Enterobacteriaceae species has shown the wide distribution of multidrug-resistant C. freundii, including ST22 strains with close evolutionary distance.⁴¹

Overall, our data showed the first detection of NDM-1 and VIM-48 coproducing C. freundii strain isolated from the hospital environment in Tunisia and the second detection in the world of the bla_VIM-48 gene. The presence of an extremely multidrug-resistant strain coharboring two MBL genes and containing various plasmid types in the hospital environment showed a higher risk of large-scale propagation of multiresistant bacteria constituting, therefore, a real danger to public health.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by the Tunisian Ministry of Higher Education and Scientific Research, the Military Hospital of Tunisia, and the University-Cerrahpaşa of Istanbul, Turkey.

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